Avian influenza (AI) is a viral disease widely affecting the poultry industry across the world. Between 1877 and 1958 several epizootics were reported to be caused by highly pathogenic viruses in most regions of the world (1,2); within following years, 26 outbreaks of the infection occurred, although the losses were insignificant (3,4,). Until 2008, four foci alone affected several millions of birds (5.6).
The most recent outbreaks were outshined by the panzootic of the H5N1 AI virus subtype which spread across Asia, Europe and Africa, affecting more than 60 countries and causing the death of hundreds of millions of birds (7).
Controlling the situation became rather difficult. International organizations implemented immediate and direct in-field actions (8.9), while the World Organization for Animal Health (WOAH) established emergency vaccination as an important tool when an outbreak is detected (10). The effectiveness of oil-adjuvant inactivated vaccines in the reduction of AI mortality and/or the prevention of the disease in chickens and turkeys (11, 12) has been demonstrated.
In order to face emergency vaccination, a vaccine strain bank should be previously contemplated in emergency plans against the disease (10,13), and an organizational system based in the creation of primary and secondary seed banks providing for a reference bank and a work bank is required. (14). By creating seed banks for the AI vaccine, the critical points in manufacturing a vaccine candidate is covered, therefore, obtaining and maintaining them will guarantee optimal results (15) .
Industrial and retail poultry farming in our continent has not been affected by the Asian H5N1 highly pathogenic AI (HPAI) virus thanks to sanitary measures implemented so far. However, it is important to bear in mind that countries like Canada, US, Mexico, Guatemala, El Salvador, Chile, Colombia and more recently the Dominican Republic have been affected by H5 and H7 strains of American lineage causing severe outbreaks and epizootics that have brought serious economic losses since the beginning of the 1980’s to the present (16).
Although AI is not present in Cuba, a vaccine against the disease should be produced in order to be available to face an emergency early enough; for such reason this project seeks to obtain master and work seed viruses (MSV and WSV) from a characterized AI strain as the first step in the manufacturing process of a vaccine candidate.
Materials and methods
Strain: The Ck/It/9097/97 strain isolated during the epidemiological surveillance for the control of eight HPAI outbreaks caused by the H5N2 virus was used. The isolation was made on healthy fowls from farms maintaining commercial relations with others infected with HPAI outbreaks, north of Italy [22, 23].
Multiplying or amplifying the virus strain: the strain was multiplied in chicken embryos free of specific pathogens (FSP), nine days of incubation, inoculated via allantoic cavity and incubated for 72 Hr at 37 ºC. Dead embryos at 24 Hr post-inoculation (PI) were disposed of.
Prior to making the collection, one drop of the allantoic liquid (AL) from each of the inoculated embryos were mixed individually with one drop of 10% chicken erythrocyte moderately agitated and observed during five minutes for the presence of hemagglutination virus by fast hemagglutination [24,25]. Only the embryos with positive results were collected.
Determination of infective titer of virus strain: once the viral suspension was obtained, it was tittered in chicken embryos FSP nine days old, inoculated via AC with 0.2 mL of the logarithmic dilutions and seven embryos were not inoculated and left as controls. All inoculated embryos were incubated at 37 ºC for five days.
The embryos that died within the first 24 Hr were disposed of and those that died in the following hours were preserved at 4oC for 24Hr. Each embryo was evaluated by fast hemagglutination [24, 25]. The calculation of the ineffective titer was made by the Spearman-Karber method .
Determination of hemagglutination titer of virus strain: the viral concentration was determined by means of the hemagglutination assay (HA) in U-bottom micro plaques compared with chicken erythrocytes. The work volume was 50 μL and three replicas were made. The same volume of 1% chicken red blood cells was added to the base-2 serial dilutions in PBS. The plaque was kept at room temperature for 30 min. As HA titer we considered the reciprocal to the last dilution in which complete hemagglutination was observed .
Obtaining MSV: two passages in chicken embryos were performed from the biomass obtained from the multiplication of the original lyophilized strain. The second passage, which was the MSV, was characterized in terms of identity, pathogenicity, ineffective titer, hemagglutination, microbiological and virological pureness, as established by the OIE (2008)  and the CFR (2006) .
Identity: it was determined by means of the inhibition hemagglutination technique (IHA)  in U-micro plaques and the beta method, constant virus, variable serum. Dilutions of all 16 AI Hemagglutinin antiserums and the specific 1 Paramyxovirus antiserum were used. The hemagglutination inhibiting titer was determined with the last antiserum dilution in which complete inhibition of hemagglutination activity was observed.
The neuraminidase (N) inhibition was performed according to reports by Capua and col., 2003 . Antiserums with H different from the virus to be evaluated for the nine N subtypes were selected. The neuraminidase activity inhibition was determined by the observation of white or slightly yellow coloring in the tubes as compared with the control virus. Pink coloring indicated non neuraminidase inhibition.
Pathogenic characterization of the biomass: the intravenous pathogenicity index value (IPIV) was determined in 10 six-week-old chicken FSP previously inoculated via intravenous with 0.1 mL of a 1:10 MSV dilution. The IPIV was calculated as mean per chicken based on observation during 10 days . The results were interpreted according to OIE (2008) .
MSV microbiological and virological pureness
a) Detection of bacterial and mycotic contaminating agents: the fungal and bacterial contamination of the MSV was determined according to the CFR (2006)  and the European Pharmacopeia (2005) . Inoculation was made by direct planting in tubes with culture means of thioglycolate and soy tripthone culture medium, that were later incubated at 35 ºC to 37 ºC and 20 ºC- 25 ºC respectively under daily observation for 14 days. The absence of cloudiness or formation of films in the planted mediums indicated that the problem sample satisfies the essay.
b) The contamination by mycoplasma was determined according to CFR (2006)  by means of the following methods: planting in blood agar, culture planting for mycoplasmas, crossbreeding of nucleic acids and polymerase chain reaction (PCR). Mycoplasma arginini and Mycoplasma gallisepticum were used as reference materials.
c) Detection of viral contaminating agents.
- Chicken embryos: an aliquot part of MSV was neutralized with nine parts of its specific reference antiserum and it was incubated at 37°C for 30 minutes. Subsequently, 0.2 mL of the resulting mixture was inoculated in:
• 10 FSP embryos (10 days) by chorioallantoic membrane (CAM).
• 10 FSP embryos (10 days) by allantoic cavity (CA).
• 10 FSP embryos (6 days) by vitelline sac.
All the embryos were incubated during seven days at 37°C and daily observation was made by ovoscopy.
The embryos that died within the first 24 Hr were disposed of and those that died after that period were evaluated by fast HA, as previously described. The CAMs were also examined for lesions. The embryos that survived the seventh day were also examined. The embryos inoculated via vitelline sac were incubated and observed on a daily basis until hatching. The chicks were observed for 10 days.
- Cell cultures: culture bottles with converging monolayers of chicken embryo fibroblast cells (CEFC) from FSP chicken embryos and VERO cell lines were inoculated each with 1 mL of MSV neutralized with its specific antiserum. Control bottles of both cells were submitted to the same treatment but with PBS as inoculating substance. All inoculated cells were incubated at 37°C for 45 min. Then maintenance medium was added and they were incubated at 37 °C for five days under daily observation. Three blind passages were performed every five to seven days with three replicas each. Each passage was frozen and defrosted three times to inoculate the following one.
At the end of the incubation period of the third passage of both inoculated cell groups the maintenance medium was removed from one of the replicas and from one control. The monolayers were washed with PBS. Next, a 1% PBS suspension of chicken red blood cells (v/v) was added to each of the bottles and they were incubated at 4°C for 30 min . They were washed with PBS and examined on inverted-lens microscope for hemadsorption.
The remaining culture bottles were washed with PBS, the cells were fixed with methanol, dried and then dyed with giemsa 1:15, during 20 min. They were washed with deionized and were observed on the microscope in search of inclusion bodies, abnormal number of giant cells or any other alterations that may be associated to contaminating agents.
- Chickens: 10 FSP chickens were used, 2-3 weeks old, previously inoculates via intramuscular (IM) and intraocular (IO) with 100 and 50 doses respectively of MSV neutralized with its specific antiserum. On day 14 PI the animals received a second inoculation be the same means with same amount doses. 10 chickens were left out as controls without being inoculated and blood samples were taken before the first and second inoculations and at day 42 PI. The serums of sampled animals in the different timings were evaluated by ELISA (IDEXX) for the determination of antibodies to avian infectious anemia virus, reticuloendotheliosis, avian infectious bronchitis, gumboro and avian leucosis.
Multiplication of MSV to obtain WSV: in order to obtain the WSV two passages were performed in chicken embryos starting from the MSV obtained. The WSV was evaluated regarding ineffective titer, hemagglutination titer, microbiological and virological pureness, as described for MSV, according to the OIE (2008a)  and the CFR (2006) .
Results and discussion
The Ck/It/9097/97 strain, low pathogenicity H5N9 AI virus has been used in different countries and regions (Italy, Mexico, Central America and Asia) for the development of vaccines effective in the control of AI outbreaks [32-34].
From multiplying the original strain in FSP chicken embryos a viral biomass with ineffective titer of 107.3 DIE50/mL and hemagglutination titer of 1/1024 was obtained.
When comparing the results with the titers developed by the AI isolates Potsdam/86 and Mexico/94 (105.1 and 105.9 DIE50/mL, respectively), which are used in internationally recognized vaccines , we can state that the analyzed strain showed a highly-ineffective titer.
The MSV virus was established on the second passage, a stage close to the original strain, to prevent the possible loss of its properties due to successive passages.
In the determination of the MSV identity, the IHA test produced a positive reaction of hemagglutination inhibition to the H5 antiserum. Hemagglutination inhibition was not produced with the remaining reference IA specific antiserums, neither with the Avian Paramyxovirus Type 1 (Newcastle) specific antiserum. In the case of N inhibition (INA), the analyzed sample showed a white, yellowish color to the N9 antiserum, which demonstrates that the enzymatic activity of N was inhibited by the antiserum. Such results confirm that the MSV obtained matches the identity of the H5N9 original strain taken as starting point.
The result of the IPIV for the pathogenic characterization was 0.00. Neither clinical signs nor mortality from infection with the AI virus were observed in the inoculated chickens during the assay. This results match reports by Capua and col. (1999)  and by OIE (2008)  for low pathogenicity AI strains (LPAI).
The ineffective titer results of the MSV and the WSV (108.5 and 109.1 DIE50/mL) are higher than the amount of virus needed to cause hemagglutination (105-106 DIE50/mL); this is key feature in working with this virus . The WSV ineffective titer matches references by the Mexican official standards  for the manufacturing of emulsified inactivated vaccines against H5N2 AI stating that the minimum titer for WSV should be between 108.7 and 109 DIE50/mL.
The evaluated samples of MSV and WSD were negative to bacterial and fungal control in the plantings made on the different culture mediums during 14 days of incubation, therefore, they were considered as sterile. The samples analyzed were also negative to the detection of mycoplasmas in the three assays used.
During the search of contaminating agents in FSP chicken embryos inoculated via CAM, CA and VS with neutralized MSV and WSV did not show membrane lesions like swelling of the presence of foci. The AL evaluated did not show reactions of hemagglutination or malformations in the chicken embryos that could show the presence of contaminating viruses. (table 1).
Table 1. Quality Controls performed on MSV and WSV.
108,5 DIE 50/ml
Contaminating viral agents in chicken embryos
Contaminating viral agents in cell cultures of FSP and VERO
Contaminating viral agents in chickens
Negative to all evaluated entities
Negative to all evaluated entities
In the search for contaminating agents in chickens for the MSV and WSV neither respiratory symptoms nor nervous symptoms nor others of any kind were found. All the serum samples studied were negative to avian infectious anemia, reticuloendotheliosis, gumboro, avian infectious bronchitis, avian encephalomyelitis, reovirus and avian leucosis (table 1).
The results of the three serial inoculations on cell cultures of FSP and VERO cell lines did not yield cytopathy effects, otinclusion bodies, giant cells, or visible hemadsorption or other cell modification indicating contamination with other viral agents (table 1). These results demonstrate that during the process of obtaining the MSV and WSV there was no contamination with contaminating agents and that it fulfills the standards established by the CFR (2006)  and the OIE (2008)  for it use in vaccine manufacturing.
Biological productions are made from the MSV and WSV evaluated as satisfactory in microbiology and virology pureness tests . The results of this study show that the seed obtained is reliable. Its bacteriological pureness was demonstrated as well as the absence of contaminating virus, hemagglutinating or not, which could bring about misleading results.
For any vaccine to be efficient, the Hemagglutinin of the selected strain must be antigenically related with the circulating strain (field) to ensure that the antibodies produced by the vaccine strain are able to neutralize the field virus [39,40]. This way the release of viral particles can be remarkably controlled and reduced. In addition, vaccines against H5 AI for chicken have been proved to be effective in inducing protection for many years against isolated H5 viruses. [41, 42].
The strain used in this study Ck/It/9097/97, H5N9 has a subtype of Hemagglutinin that is equivalent to the one currently circulating in Asia (H5), Africa and Middle East and represents a threat to our continent, either because of the migrating cycles of birds coming from those regions or due to the international movement of poultry or risk products. Also, it demonstrated in the assays a good multiplication (high ineffective titers) in embryo eggs of FSP chicken.
The WSV obtained comes from this low pathogenicity strain, demonstrated by the pathogenic characterization study carried out in Cuba, coming from the OIE Reference Laboratory for the diagnosis of AI and Newcastle in Venice; therefore, we fulfill the OIE (2008a) requirements ] to establish seeds for the manufacturing of inactivated vaccines.
The MSV and WSB was obtained from the AI H5N9 strain meeting quality standards regarding identity, pathogenicity, infective titer, HA titer and microbiological and virological pureness.
The possibility of having MSV and WSV available which ensure the quality and quantity required for manufacturing the vaccine is an important strategic and economic element to our country, as they will avoid importing the vaccine in case of AI outbreaks, as well as the possible biological risk of introducing non-circulating strains in the country.
The MSV and WSV obtained from AILP strain meet the quality standards established for the manufacturing of inactivated vaccines against that disease.
To researches of the Center for Scientific Research of the Civil Defense Council (CICDC), of the National Center for Agricultural Livestock Health (CENSA) and the State Control laboratory of the Institute of Veterinary Medicine (IMV) who collaborated, one way or another, in the realization of this work.
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La avicultura industrial y artesanal en nuestro continente, debido a todas las medidas sanitarias tomadas hasta el momento, no se ha visto afectada por el virus de la IA altamente patógena (IAAP) H5N1 del subtipo asiático.